DNA sequencing as practiced today involves a series of steps starting from isolating DNA from the biological sample, cutting these into fragments with convenient sizes, amplifying the fragments biochemically, introducing a label for detection while generating a nested set of ordered fragments, separating the ordered set of fragments and identifying the nucleotide sequence, and reassembling the short sequence data into a continuous sequence. Recent developments in capillary and in microchannel electrophoresis, especially in multiplexed arrays, show great promise for substantially increasing the speed and throughput of the separation and identification steps. The issue of cost and compatibility with the other steps in the whole sequencing process still remains. This proposal is aimed towards the development of novel front-end technologies, whereby the speed and throughput of sample preparation can be significantly increased while the amount of manual operation and total reagent and instrumental costs can be significantly reduced. These technologies will specifically take advantage of the inherent suitability of capillaries and microchannels for utilizing small samples and for highly multiplexed operation during the subsequent separation and detection steps. Several distinct goals can be identified. 1. Demonstration of the production of fluorescently-labeled Sanger fragments via cycle-sequencing in capillary tubes in total volumes of 10- 100 nL. This will lead to the consumption of 100-fold less sample, enzymes, fluorescent labels and other reagents. 2. Development of automated microfluidics to achieve simultaneous multiplexed operation of cycle-sequencing in 96 capillary tubes. This will allow one-step one-line operation starting with the injection of 10-100 nL of each template to provide nucleotide sequence identification in a capillary array electrophoresis system. 3. Development of direct sequencing from a bacterial colony without amplification by re-incubation and without separate extraction and pre- purification steps.
